Four single-basepair mutations in the ptx promoter of Bordetella bronchiseptica are sufficient to activate the expression of pertussis toxin.

Secretion of pertussis toxin (PT) is the preeminent virulence trait of the human pathogen Bordetella pertussis, causing whooping cough. Bordetella bronchiseptica, although it harbors an intact 12-kb ptx-ptl operon, does not express PT due to an inactive ptx promoter (Pptx), which contains 18 SNPs (single nucleotide polymorphisms) relative to B. pertussis Pptx. A systematic analysis of these SNPs was undertaken to define the degree of mutational divergence necessary to activate B. bronchiseptica Pptx. A single change (C-13T), which created a better - 10 element, was capable of activating B. bronchiseptica Pptx sufficiently to allow secretion of low but measureable levels of active PT. Three additional changes in the BvgA-binding region, only in the context of C-13T mutant, raised the expression of PT to B. pertussis levels. These results illuminate a logical evolutionary pathway for acquisition of this key virulence trait in the evolution of B. pertussis from a B. bronchiseptica-like common ancestor.

A case of cervical subcutaneous abscess due to Bordetella hinzii.

We present a case of subcutaneous infection caused by Bordetella hinzii in a healthy male. The isolate was successfully identified by gyrB gene sequencing. B. hinzii cannot be distinctively identified using 16S rRNA gene sequencing or by biochemical methods. The number of cases infected with B. hinzii might be underestimated owing to the difficulty in accurate identification, which can be achieved by gyrB gene sequencing to gain knowledge about the species.

Subcutaneous Immunization of Dogs With Bordetella bronchiseptica Bacterial Ghost Vaccine.

The Bordetella species are Gram-negative bacterial pathogens that colonizes mammalian respiratory tract causing respiratory diseases in humans and animals. B. bronchiseptica causes clinical conditions in many mammals including immunocompromised humans. Using the dog model of respiratory infection, it has been shown in this study that a newly developed B. bronchiseptica Bacterial Ghost (BbBG) vaccine exhibited significant protection in the face of a severe pathogenic bacterial challenge in seronegative dogs. The protein E-specific lysis mechanism was used to produce BbBGs. Bacterial Ghosts (BGs) are the empty cell envelope of Gram-negative bacterium. They are genetically processed to form a microscopic hole in their membrane, through which all the cytoplasmic contents are expelled leaving behind intact empty bacterial shells. Due to the intact surface structures of BGs, they offer the safety of inactivated but efficacy of live attenuated vaccines. In this study, seronegative dogs were vaccinated subcutaneously (s/c) with two different doses of a newly developed BbBG vaccine [lower 10∧5 (BbBG - 5) and higher 10∧7 (BbBG - 7)] on day 0 and 21. The animals were challenged (by aerosol) with virulent live B. bronchiseptica strains 41 days after first vaccination. The dogs vaccinated s/c with BbBG - 7 vaccine had significantly lower spontaneous coughing scores (P = 0.0001) than dogs in negative control group. Furthermore, the tested BbBG - 7 vaccine was equivalent to the positive control vaccine Bronchicine CAe in terms of safety and efficacy. For the first time, we report the successful use of liquid formulated BGs vaccines in animal studies. Earlier reported studies using BGs vaccines were performed with resuspended freeze-dried BGs preparations.

FOXA2 depletion leads to mucus hypersecretion in canine airways with respiratory diseases.

Because of exposure to environmental pollutants, infectious agents, and genetic predisposition, companion animals develop respiratory illnesses similar to those in humans. Older dogs of smaller breeds develop canine infectious respiratory disease, chronic bronchitis, and chronic obstructive pulmonary disease, with chronic lung infection, airway goblet cell hyperplasia and metaplasia, and mucus hypersecretion. Excessive mucus clogs airways, reduces gas exchanges, disables the mucociliary clearance, and reduces drug penetration. The Forkhead box protein A2 (FOXA2) is a key transcriptional regulator that maintains airway mucus homeostasis. Prior studies have shown that FOXA2 expression is frequently depleted in diseased human airways. Unfortunately, FOXA2 depletion has not been examined in dogs. Our current study indicated that both single bacterial infection by Pseudomonas aeruginosa and Bordetella bronchiseptica and polymicrobial infection by viral/bacterial pathogens depleted FOXA2 in canine airways, resulting in goblet cell hyperplasia and metaplasia and excessive mucus production. Furthermore, P. aeruginosa virulence factor pyocyanin activated the antagonistic STAT6 and epidermal growth factor receptor signalling pathways to inhibit FOXA2. Unravelling the mechanism of FOXA2 inactivation will hasten the development of non-antibiotic therapeutics to improve mucociliary clearance of pathogens in canine airway.

Immunomodulation as a Novel Strategy for Prevention and Treatment of Bordetella spp. Infections.

Well-adapted pathogens have evolved to survive the many challenges of a robust immune response. Defending against all host antimicrobials simultaneously would be exceedingly difficult, if not impossible, so many co-evolved organisms utilize immunomodulatory tools to subvert, distract, and/or evade the host immune response. Bordetella spp. present many examples of the diversity of immunomodulators and an exceptional experimental system in which to study them. Recent advances in this experimental system suggest strategies for interventions that tweak immunity to disrupt bacterial immunomodulation, engaging more effective host immunity to better prevent and treat infections. Here we review advances in the understanding of respiratory pathogens, with special focus on Bordetella spp., and prospects for the use of immune-stimulatory interventions in the prevention and treatment of infection.

Development of macrolide resistance in Bordetella bronchiseptica is associated with the loss of virulence.

Background: Why resistance to specific antibiotics emerges and spreads rapidly in some bacteria confronting these drugs but not others remains a mystery. Resistance to erythromycin in the respiratory pathogens Staphylococcus aureus and Streptococcus pneumoniae emerged rapidly and increased problematically. However, resistance is uncommon amongst the classic Bordetella species despite infections being treated with this macrolide for decades. Objectives: We examined whether the apparent progenitor of the classic Bordetella spp., Bordetella bronchiseptica, is able to rapidly generate de novo resistance to antibiotics and, if so, why such resistance might not persist and propagate. Methods: Independent strains of B. bronchiseptica resistant to erythromycin were generated in vitro by successively passaging them in increasing subinhibitory concentrations of this macrolide. Resistant mutants obtained were evaluated for their capacity to infect mice, and for other virulence properties including adherence, cytotoxicity and induction of cytokines. Results: B. bronchiseptica rapidly developed stable and persistent antibiotic resistance de novo. Unlike the previously reported trade-off in fitness, multiple independent resistant mutants were not defective in their rates of growth in vitro but were consistently defective in colonizing mice and lost a variety of virulence phenotypes. These changes rendered them avirulent but phenotypically similar to the previously described growth phase associated with the ability to survive in soil, water and/or other extra-mammalian environments. Conclusions: These observations raise the possibility that antibiotic resistance in some organisms results in trade-offs that are not quantifiable in routine measures of general fitness such as growth in vitro, but are pronounced in various aspects of infection in the natural host.

The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis.

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

Bordetella trematum sepsis with shock in a diabetic patient with rapidly developing soft tissue infection.

Bordetella is a gram-negative, glucose non-fermenting bacillus, consisting of many host-associated species. B. trematum has previously been identified in wound infections, but rarely known to be a source of bacteremia. Currently, 16S rRNA sequencing represents the reference standard method by which identification is made. Herein, we present a case of fatal B. trematum bacteremia with septic shock. The presumed primary site of the infection was a rapidly developing left leg deep soft tissue infection without necrotizing fasciitis. B. trematum should now be considered as a significant pathogen in sepsis.

Colonización crónica por Bordetella petrii. Primer caso en España / Bordetella petrii chronic colonization. First case in Spain

No disponible

The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca(2+) -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

Interspecies variations in Bordetella catecholamine receptor gene regulation and function.

Bordetella bronchiseptica can use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and although Bordetella pertussis has the bfrD and bfrE genes, the role of these genes in iron uptake has not been demonstrated. In this study, the bfrD and bfrE genes of B. pertussis were shown to be functional in B. bronchiseptica, but neither B. bronchiseptica bfrD nor bfrE imparted catecholamine utilization to B. pertussis. Gene fusion analyses found that expression of B. bronchiseptica bfrA was increased during iron starvation, as is common for iron receptor genes, but that expression of the bfrD and bfrE genes of both species was decreased during iron limitation. As shown previously for B. pertussis, bfrD expression in B. bronchiseptica was also dependent on the BvgAS virulence regulatory system; however, in contrast to the case in B. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression of bfrD in B. bronchiseptica. Further studies using a B. bronchiseptica bvgAS mutant expressing the B. pertussis bvgAS genes revealed that the interspecies differences in bfrD modulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to the in vivo fitness of B. bronchiseptica and B. pertussis. Additional evidence of the in vivo importance of the B. pertussis receptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with the B. pertussis BfrD and BfrE proteins.

Widespread Bordetella parapertussis Infections-Wisconsin, 2011-2012: Clinical and Epidemiologic Features and Antibiotic Use for Treatment and Prevention.

BACKGROUND: During October 2011-December 2012, concurrent with a statewide pertussis outbreak, 443 Bordetella parapertussis infections were reported among Wisconsin residents. We examined clinical features of patients with parapertussis and the effect of antibiotic use for treatment and prevention. METHODS: Patients with polymerase chain reaction results positive for B. parapertussis reported during October 2011-May 2012 were interviewed regarding presence and durations of pertussis-like symptoms and receipt of azithromycin treatment. Data regarding acute cough illnesses and receipt of azithromycin prophylaxis among parapertussis patient household members (HHMs) were also collected. Using multivariate repeated measures log-binomial regression analysis, we examined associations of treatment receipt by the HHM with the earliest illness onset and prophylaxis receipt among other HHMs with the presence of any secondary cough illnesses in the household. RESULTS: Among 218 patients with parapertussis, pertussis-like symptoms were frequently reported. Illness durations were significantly shorter among patients with treatment initiated 0-6 days after cough onset, compared with nonrecipients (median durations: 10 vs 19 days, P = .002). Among 361 HHMs from 120 households, compared with nonrecipients, prompt prophylaxis of HHMs was associated with no secondary cough illnesses (relative risk: 0.16; 95% confidence interval, .04-.69). CONCLUSIONS: Bordetella parapertussis infection causes pertussis-like illness that might be misclassified as pertussis if B. parapertussis testing is not performed. Prompt treatment might shorten illness duration, and prompt HHM prophylaxis might prevent secondary illnesses. Further study is needed to evaluate antibiotic effectiveness for preventing parapertussis and to determine risks and benefits of antibiotic use.

Bordetella holmesii infection: current knowledge and a vision for future research.

Bordetella holmesii is a recently recognized Gram-negative bacterium causing both pertussis-like respiratory symptoms and invasive infections, such as bacteremia, pneumonia, meningitis, arthritis, pericarditis and endocarditis. Few data are available on its epidemiological characteristics, mostly related to respiratory infections. However, these are frequently misdiagnosed as a Bordetella pertussis infection as most diagnostic tests routinely used are not species-specific, thus biasing the epidemiological studies of both strains, as well as the efficacy studies on pertussis vaccination. There is no accepted agreement on treatment and it remains unknown if antimicrobial prophylaxis is indicated in certain clinical settings. We review here the current knowledge on B. holmesii and the need for further research.

Bordetella trematum bacteremia in an infant: a cause to look for.

Bordetella trematum spp. nov. has been isolated from wounds, ear infections and diabetic ulcers. We report a case of a 7-month-old infant with fever, vomiting and abnormal body movements with bacteremia caused by this novel species. The infant responded to fluoroquinolone and macrolide combination therapy.

Isolation of Bordetella bronchiseptica from blood and a pancreatic abscess.

Bordetella bronchiseptica is a respiratory pathogen rarely encountered in human hosts. We describe a case of bacteremia and pancreatic abscess caused by this organism. To our knowledge, this is the first reported case of B. bronchiseptica causing intra-abdominal infection in the form of an abscess.

Microbiological and clinical aspects of respiratory infections associated with Bordetella bronchiseptica.

Bordetella bronchiseptica is a well-known veterinary pathogen, but its implication in human disease is probably not fully recognized. The purpose of this study was to determine the clinical significance of 36 B. bronchiseptica isolates from respiratory samples of 22 patients. Therefore, we describe microbiological characteristics, including phenotypic and genotypic identification as well as antimicrobial susceptibilities of the isolates. Clonal relatedness was evaluated using pulsed-field gel electrophoresis (PFGE). Most of the patients had some underlying immunosuppressive condition. Eighteen out of 22 (82%) patients had respiratory symptoms, and the death of 2 patients was associated with respiratory infection.All strains were correctly identified at species level by the simultaneous use of phenotypic methods and were confirmed by specific amplification of the upstream region of the fla gene. Tigecycline, minocycline, doxycycline, colistin, and meropenem were the most active agents tested. PFGE analysis revealed that repeated infections involving each patient had been caused by the same strain.

Septic arthritis and osteomyelitis due to Bordetella petrii.

A case of Bordetella petrii septic arthritis and osteomyelitis in an elbow resulted from a dirt bike accident in Hawaii. Two months of intravenous antibiotics and repeated surgeries were required to cure this infection. Our case, and literature review, suggests that extended-spectrum penicillins, tetracycline, and trimethoprim-sulfamethoxazole are good treatment options.

Caspase-1-independent interleukin-1ß is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/ß and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of Bordetella pertussis. We show that IL-1ß but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1ß and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1ß to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1ß responses that are critical for bacterial clearance and vaccine-mediated immunity.

Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection.

Worldwide resurgence of pertussis necessitates the need for improvement of pertussis vaccines and vaccination strategies. Since natural infections induce a longer-lasting immunity than vaccinations, detailed knowledge of the immune responses following natural infection can provide important clues for such improvement. The purpose was to elucidate the kinetics of the protective immune response evolving after experimental Bordetella pertussis (B. pertussis) infection in mice. Data were collected from (i) individual analyses, i.e. microarray, flow cytometry, multiplex immunoassays, and bacterial clearance; (ii) twelve time points during the infection; and (iii) different tissues involved in the immune responses, i.e. lungs, spleen and blood. Combined data revealed detailed insight in molecular and cellular sequence of events connecting different phases (innate, bridging and adaptive) of the immune response following the infection. We detected a prolonged acute phase response, broad pathogen recognition, and early gene signatures of subsequent T-cell recruitment in the lungs. Activation of particular transcription factors and specific cell markers provided insight into the time course of the transition from innate towards adaptive immune responses, which resulted in a broad spectrum of systemic antibody subclasses and splenic Th1/Th17 memory cells against B. pertussis. In addition, signatures preceding the local generation of Th1 and Th17 cells as well as IgA in the lungs, considered key elements in protection against B. pertussis, were established. In conclusion, molecular and cellular immunological processes in response to live B. pertussis infection were unraveled, which may provide guidance in selecting new vaccine candidates that should evoke local and prolonged protective immune responses.

Cytokine and chemokine mRNA expression profiles in BALF cells isolated from pigs single infected or co-infected with swine influenza virus and Bordetella bronchiseptica.

Pigs serve as a valuable animal experimental model for several respiratory pathogens, including Swine Influenza Virus (SIV) and Bordetella bronchiseptica (Bbr). To investigate the effect of SIV and Bbr coinfection on cytokine and viral RNA expression, we performed a study in which pigs were inoculated with SIV, Bbr or both pathogens (SIV/Bbr). Our results indicate that Bbr infection alters SIV clearance. Pulmonary lesions in the SIV/Bbr group were more severe when compared to SIV or Bbr groups and Bbr did not cause significant lesions. Broncho-alveolar lavage fluid (BALF) was examined for inflammatory mediators by qPCR. Interferon (IFN)-α, interleukin IL-8, IL-1 peaked in BALF at 2 DPI, while the virus titres and severity of clinical signs were maximal at the same time. Despite its increased expression in co-infected pigs, interferon-α did not enhance SIV clearance, since the viral replication was detected at the same day as the highest IFN levels. The mRNA levels for IFN-α, IL-1ß and IL-8 were significantly higher in BALF of co-infected pigs and correlated with enhanced viral RNA titers in lungs, trachea and nasal swabs. Transcription of mRNA for IL-1ß was stable in SIV and SIV/Bbr groups throughout all the study. In Bbr group, the levels of mRNAs for IL-1ß were significantly higher at 2, 4 and 9 DPI. The mean levels of mRNAs for TNF-α were lower than the levels of other chemokines and cytokines in all infected groups. Transcript levels of IL-10 and IL-4 did not increase at each time points. Overall, SIV replication was increased by Bbr presence and the enhanced production of pro-inflammatory mediators could contribute to the exacerbated pulmonary lesions.